Continuous multilayered composite hydrogel as osteochondral substitute.

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

4% articaine buccal infiltration versus 2% lidocaine inferior alveolar nerve block for emergency root canal treatment in mandibular molars with irreversible pulpits: a randomized clinical study.

AIM: To compare the anaesthetic efficacy of inferior alveolar nerve blocks (IANB) with 1.8 mL of 2% lidocaine (LI) to a buccal infiltration (BI) with 1.8 mL of 4% articaine (AR), both with 1 : 100 000 epinephrine, in patients with symptomatic irreversible pulpits in a randomized controlled trial. METHODOLOGY: Volunteers presenting at the Emergency Centre (FOP-UNICAMP) were randomly divided into two groups (30 for AR and 20 for LI). Operator and patient were not blinded. Success was recorded when complete pain-free treatment was achieved after a single injection (IANB or BI) or when one supplemental injection was needed for emergency endodontic procedures. Success rate of supplemental injection was evaluated between and within groups using Fisher's exact test and chi-square test. RESULTS: A higher success rate (P = 0.03/Fisher's exact test) was observed with AR (40%) than with LI (10%). No significant difference was found when a single injection plus one supplemental injection was compared between groups (P = 1.0; AR = 70%; LI = 80%). However, supplemental injection increased the anaesthetic success rates (AR, P = 0.04; LI, P = 0.0001) within groups. CONCLUSIONS: Single anaesthesia techniques (IANB or BI) were not able to achieve pain-free emergency endodontic treatment. Supplemental anaesthetic techniques should be considered prior to treatment procedures in order to increase success rate (consort: registration number - NCT01912755/Fapesp: #2009/10834-4).

Anaesthetic efficacy of bupivacaine 2-hydroxypropyl-ß-cyclodextrin for dental anaesthesia after inferior alveolar nerve block in rats.

Bupivacaine is a long-acting local anaesthetic that is widely used in medicine and dentistry. The duration and intensity of its sensory blockade in animal models is increased by its inclusion in complexes with cyclodextrins. The aim of the present study was to evaluate the anaesthetic efficacy of bupivacaine 2-hydroxypropyl-ß-cyclodextrin (HPßCD) inclusion complex for dental anaesthesia after inferior alveolar nerve block in rats. Thirty rats were each given an injection close to the mandibular foramen of 0.2ml of one of the following formulations: 0.5% bupivacaine alone; 0.5% bupivacaine with 1:200,000 epinephrine; and 0.5% bupivacaine-HPßCD inclusion complex (bupivacaine-HPßCD). The other sides were used as controls, with either 0.9% saline or anaesthetic-free HPßCD solution being injected. The onset, success, and duration of pulpal anaesthesia were assessed by electrical stimulation ("pulp tester") on inferior molars. Results were analysed using ANOVA (Tukey), log rank, and chi square tests (α=5%). There were no differences among the formulations in onset of anaesthesia (p=0.59) or between the bupivacaine plus epinephrine and bupivacaine plus HPßCD in duration of anaesthesia, but bupivacaine plus epinephrine gave significantly higher values than bupivacaine alone (p=0.007). Bupivacaine plus epinephrine was a better anaesthetic than bupivacaine alone (p=0.02), while Bupi-HPßCD gave intermediate results, and therefore did not differ significantly from the other 2 groups (p=0.18 with bupivacaine alone; and p=0.44 with bupivacaine plus epinephrine). The bupivacaine-HPßCD complex showed similar anaesthetic properties to those of bupivacaine with epinephrine.

Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility.

BACKGROUND AND PURPOSE: The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACH: A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 10(6) MC-26 cells (n= 16 each group). KEY RESULTS: Treatment with 5% (w w(-1)) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE(2) levels (with concomitant increased production of PGE(3)). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50-200 µM) in vitro was rescued by exogenous PGE(2) (10 µM) and PGE(1)-alcohol (1 µM). CONCLUSIONS AND IMPLICATIONS: EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a 'PGE(2) to PGE(3) switch' in liver tumours. Inhibition of PGE(2)-EP(4) receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.

Efficacy of liposome-encapsulated 0.5% ropivacaine in maxillary dental anaesthesia.

The aim of this study was to evaluate the effectiveness of liposome-encapsulated ropivacaine (0.5%) in dental anaesthesia. This randomised, double-blind, crossover, four-period treatment study included 40 volunteers who were given 1.8 ml of the following local anaesthetics into the buccal sulcus at the right level of the upper canine: 0.5% ropivacaine; 0.5% ropivacaine with 1:200,000 adrenaline; liposome-encapsulated 0.5% ropivacaine; and 2% lignocaine with 1:100,000 adrenaline. Onset of pulpal anaesthesia; the success of anaesthesia; and the duration of labial, gingival, and pulpal anaesthesia involving the upper right canine and first premolar were evaluated. At the end of each injection, volunteers rated the pain on injection on a visual analogue scale (VAS). Both ropivacaine and adrenaline, and lignocaine with adrenaline, were more successful anaesthetic agents than liposome-encapsulated ropivacaine or plain ropivacaine (p<0.05). There were no significant differences among the anaesthetic preparations in the onset of pulpal anaesthesia. Ropivacaine and adrenaline and lignocaine and adrenaline gave a significantly longer duration of pulpal anaesthesia. VAS showed no significant differences among the groups tested. The results showed that encapsulation of liposome did not improve the anaesthetic efficacy of ropivacaine.

Liposomal delivery system for topical anaesthesia of the palatal mucosa.

An effective topical agent to reduce pain during local anaesthesia of the palate is not yet available. The aim of the present study was to evaluate the efficiency of liposome-encapsulated ropivacaine in different concentrations for topical anaesthesia of the palatal mucosa. In this single-blinded, placebo-controlled, crossover study 40 (20 male) healthy volunteers were randomised to be given: liposome-encapsulated 2% ropivacaine, liposome-encapsulated 1% ropivacaine, a eutectic mixture of 2.5% lidocaine and 2.5% prilocaine (EMLA), and liposomal placebo gel, topically on to the palatal mucosa of the right canine region for 5 min each, at four different sessions. Pain associated with insertion of a 30G needle, and with injection of a local anaesthetic, was rated on a visual analogue scale (VAS). The effect of liposomal ropivacaine 1% and 2% did not differ from that of placebo (p=0.3 and p=0.1, respectively) in reducing pain during insertion of the needle. Lower VAS were obtained with EMLA. In this group VAS were lower in women than men (p=0.007). There was no difference in VAS among groups (p=0.3) as far as injection of the local anaesthetic was concerned. In conclusion, liposomal-encapsulated ropivacaine formulations did not reduce the pain of insertion of a needle into the palatal mucosa. None of the anaesthetic formulations tested, including the positive control (EMLA), were effective in reducing the pain of an injection of local anaesthetic compared with placebo.

Morphological changes in the position of the mandibular foramen in dentate and edentate Brazilian subjects.

This study assessed the mandibular foramen (MF) position variability in dentate and edentate Brazilian mandibles. Eighty dentate and 79 edentate mandibles of unknown sex were measured bilaterally using a digital caliper (0.1-mm precision). Horizontal linear measurements (HM) were done from the MF to the anterior border of the mandibular ramus (MF-A) and from the MF to the posterior border of the mandibular ramus (MF-B). Vertical linear measurements (VM) were done from the MF to the most inferior point of the mandibular notch (MF-C) and from the MF to the inferior border of the mandibular ramus (MF-D). Data were analyzed by two-way ANOVA (alpha = 5%). The HM means and standard deviations (+/-SD) for MF-A were, edentate right (ER): 17.5 (+/-3.2) mm, edentate left (EL): 17.4 (+/-3.4) mm, dentate right (DR): 19.2 (+/-3.6) mm, and dentate left (DL): 18.8 (+/-3.8) mm. The means (+/-SD) for the MF-B measurements were, respectively, ER: 12.8 (+/-2.4) mm, EL: 12.9 (+/-2.3) mm, DR: 14.2 (+/-2.4) mm, and DL: 13.9 (+/-2.6) mm. The VM values for the MF-C measurements were, ER: 23.4 (+/-3.8) mm, EL: 22.9 (+/-3.7) mm, DR: 23.6 (+/-3.1) mm, and DL: 23.1 (+/-3) mm, and for the MF-D measurements, ER: 26.4 (+/-4.2) mm, EL: 26.4 (+/-4) mm, DR 28.3 (+/-3.9) mm, and DL 28 (+/-3.8) mm. Side had no influence (p>0.05) on any edentate or dentate mandible measurement. Dentate mandible measurements showed statistically significant differences compared to the edentate mandibles, except for MF-C. The mandibular foramen position changes with loss of teeth and this variability may be responsible for occasional failure of inferior alveolar nerve block.

Ulceration of gingival mucosa after topical application of EMLA: report of four cases.

This study reports four cases of mucosa ulceration after a 30-minute application of EMLA (0.3 g) as a topical anaesthetic in dentistry. The subjects returned the next day with a white ulceration and desquamation on the application site. EMLA cream should not be applied to the oral mucosa for 30 minutes.

Articaine and lignocaine efficiency in infiltration anaesthesia: a pilot study.

AIM: The purpose of this study was to evaluate the onset of action of pulpal and soft tissue anaesthesia, and pain experience after buccal and palatal infiltrative injections with 4% articaine with 1:100,000 adrenaline, and 2% lignocaine with 1:100,000 adrenaline. METHOD: A double blind cross-over study was conducted with 20 healthy adult subjects who, in two appointments at least two weeks apart, randomly received an infiltration anaesthesia with the solutions in the buccal and palatal regions of the upper right canine. The tooth was tested with a pulp tester before (to establish its baseline response), and after the injection, until return to the base threshold level. The pain experience caused by palatal injection was verified by the visual analogue scale (VAS). Data were analysed using Wilcoxons test (alpha = 0.05). RESULTS: There were no significant statistical differences between the solutions with respect to VAS (p = 0.45), onset of action (p = 0.80) and pulpal (p = 0.08) and soft tissue (p = 0.18) anaesthesia duration, although pulpal anaesthesia may have reached statistical significance if a higher number of volunteers had been used. CONCLUSION: Under the conditions of this study it can be concluded that both anaesthetic solutions showed similar pain experience.

Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action.

In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

Autoimmunity in isolated Addison's disease and in polyglandular autoimmune diseases type 1, 2 and 4.

Sera from 300 Italian patients with Addison's disease were collected over a 30 year period. Among these patients, 82% had autoimmune disease, 13% had tuberculosis and 5% had another causal condition. In 59% of the cases, autoimmune disease was associated with the autoimmune manifestations contributing to the description of polyglandular autoimmune disease (PGAD). In PGAD type 1, the disease was associated with chronic candidiasis and/or chronic hypoparathyroidism. In PGAD type 2, the patients had autoimmune thyroid disease and/or diabetes mellitus type 1, and in PGAD type 4, they presented a combination with other autoimmune diseases excluding those previously mentioned. Finally, the autoimmune disease was apparently isolated in 41% of the cases. In addition, patients with these four forms of disease exhibited a different genetic pattern, sex distribution, and age at presentation in addition to minor frequency of autoimmune diseases. Adrenal cortex autoantibodies directed against 21-hydroxylase were common serological markers for these four main clinical forms, showing a very high frequency at clinical onset of adrenal insufficiency. In some patients, steroid-producing cell autoantibodies were also present and correlated with gonadal failure and they recognize of 17alpha-hydroxylase or P450 side chain cleavage enzymes as target antigens.

Effects of caffeine and theophylline on the development of dental caries in rats.

The purpose of this study was to evaluate the influence of caffeine and theophylline on the development of dental caries in rats. Six Wistar dams (spf), mutans streptococci free, were obtained, each with six male pups. The dams were infected by Streptococcus sobrinus 6715 and divided into three groups which received during the lactating period: (1) diet 2000; (2) diet 2000 plus caffeine (2 mg/100 g) and (3) diet 2000 plus theophylline (0.57 mg/100 g). After weaning, the pups were infected by S. sobrinus, placed in a König-Höfer programmed feeder machine, and received 17 meals daily at hourly intervals, for five weeks. During this time the pups were fed with the same diet that their dams were. The percentage of S. sobrinus relative to total flora was significantly higher in the theophylline group. The results for slight (Ds) and moderate (Dm) dentine lesions, for smooth-surface and sulcal scores were statistically higher for the theophylline group than the other groups. Salivary assays did not demonstrate significant inorganic alterations in salivary composition. Caffeine and theophylline groups showed the highest ulcer score. It is concluded that caffeine does not affect the cariogenic potential of the diet, however theophylline can increase the development of dental caries, and this effect may be related to organic alterations of salivary composition.

Allergic reaction after rubber dam placement.

In the last few years allergic reactions to natural rubber latex (NRL) have increased in dental practice affecting both the dental team and patients. Some case reports discuss the potential risks of hypersensitivity to NRL products. An adverse patient reaction after dental rubber dam placement is reported. About 1 min after the isolation of the tooth with a rubber dam the patient presented signs and symptoms of hypersensitivity. Oxygen and intravenous hydrocortisone were administered and the patient kept under observation. After 2 h she had stable vital signs and no more allergics symptoms. It is unclear whether components of the NRL dam or the cornstarch powder incorporated with the rubber dam was responsible for the allergic reaction. Dentists must be aware of the health problem and be prepared for an adequate management in dental practice.

Adrenal-cortex autoantibodies and steroid-producing cells autoantibodies in patients with Addison's disease: comparison of immunofluorescence and immunoprecipitation assays.

Autoimmune Addison's disease and premature ovarian failure are characterized by the presence of organ-specific autoantibodies. The main adrenal and gonadal autoantigens have been identified and cloned, and the relationship between the autoantibodies detected by immunofluorescence techniques and those detected by the new assays using recombinant autoantigens needed to be investigated. We studied 165 patients with Addison's disease: 143 patients had different forms of autoimmune Addison's disease (13 with idiopathic premature ovarian failure) and 22 had nonautoimmune Addison's disease. Adrenal-cortex autoantibodies and steroid-producing cell autoantibodies were measured by the immunofluorescence techniques. Autoantibodies to steroid 21-hydroxylase, 17alpha-hydroxylase, and P450 side chain cleavage enzyme were measured by immunoprecipitation assay using 35S-labeled recombinant proteins. Adrenal-cortex autoantibodies and autoantibodies to 21-hydroxylase were found in 81% of the patients with autoimmune Addison's disease. None of the patients with nonautoimmune Addison's disease had adrenal-cortex autoantibodies or autoantibodies to 21-hydroxylase. A high association between these two markers in patients with different forms of autoimmune Addison's disease and in those with short- or long-standing disease was found. Steroid-producing cells autoantibodies were found in 26% of the patients with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage enzyme in 36% of the patients. Steroid-producing cells autoantibodies were found in 11/13 (85%) of patients with idiopathic premature ovarian failure associated with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage were found 12/13 (92%) of patients; the only case negative for all these three markers suffered from Turner's syndrome. Provided that a high standard of immunofluorescence technique is maintained, measurement of adrenal cortex autoantibodies or steroid-producing cells autoantibodies by either immunofluorescence or immunoprecipitation assay is essentially equivalent.

Acute toxicity (LD50 and CD50) of lidocaine and prilocaine in combination with adrenaline and felypressin.

The toxicity of the combination of vasoconstrictors to local anaesthetic solutions has been debated since its first use in the beginning of this century. A combination of two vasoconstrictors to a local anaesthetic has been proposed by some researchers. In this study they were evaluated the acute toxicity (lethal dose 50%, convulsion dose 50%) and latency times of loss of righting reflex and convulsion as well as the duration of convulsion) of 2% lidocaine or 3% prilocaine, when administered in combination with adrenaline and felypressin at various concentrations. Lethal dose 50% studies showed that for both anaesthetics the solutions with higher concentrations of adrenaline were more toxic. The opposite was observed in the convulsion dose 50% studies. No alterations were observed in the control groups. All lidocaine solutions increased the latency of loss of righting reflex. The latency of convulsion was increased in some groups, but once the convulsion was achieved there was no difference in its duration. There was no statistical difference among prilocaine groups for any of the variables studied. Based on the experimental model studied, it was concluded that there is no advantage in the association of two vasoconstrictors concerning the toxicity of lidocaine and prilocaine solutions.

Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies.

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.

A study of the epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison's disease.

Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH.